P347: PHARMACOLOGICAL EZRIN INHIBITION REDUCES GROWTH, ADHESION, MIGRATION AND REGULATE GENE RELATED TO APOPTOSIS AND CELL CYCLE IN ACUTE LYMPHOBLASTIC LEUKAEMIA
Authors: Jean Carlos Lipreri Da Silva, Keli Lima, Mariana Lazarini, Hugo Passos Vicari, Eduardo Magalhães Rego, Benjamin Ede, Anne Ridley, João Agostinho Machado Neto
Journal: HemaSphere (2023)

Background: The current therapeutic options available for adult patients with acute lymphoblastic leukaemia (ALL) remain limited. One of the main goals of treatment is to prevent relapses and decrease invasiveness in other organs. Recently, our research group identified the EZR gene, which encodes the protein ezrin, as an independent prognostic marker and molecular target for acute myeloid leukaemia. Ezrin is an important cytoskeleton-associated protein that promotes signal transduction between membrane proteins like tyrosine kinases, Rho GTPases, and actin filaments. The identification of new molecular markers of prognosis and signalling pathways/selective targets for pharmacological intervention may improve clinical management and generate new opportunities for therapies. Aims: The present study aimed to verify the impact of pharmacological inhibition of ezrin on viability, autonomous clonal growth, apoptosis, adhesion, migration, and gene expression in ALL. Methods: Jurkat (PTEN mutated), NALM6 (ETV6-EBF1), and REH (ETV6-RUNX1) ALL cell lines, and ALL primary cells (n=15) from bone marrow were treated with increasing concentrations of the ezrin inhibitor, NSC305787. Cell viability was evaluated by MTT, autonomous colonies by clonogenic assay, gene expression analysis by qPCR to evaluate genes related to cell cycle and apoptosis, cell adhesion evaluated by fibronectin adhesion, cell migration by transwell and CXCL12 chemotaxis, and signalling pathways were investigated by western blot and proteomic analysis. Differentially expressed protein was determined on the Galaxy tool. Statistical analyses were performed by ANOVA test and Bonferroni post-test, and a p-value <0.05 was considered statistically significant. Results: Treatment with NSC305787 significantly reduced the cell viability of Jurkat, NALM6, and REH cells in a concentration-dependent manner (p<0.05). IC50 values were 5.1, 3.3 and 4.3 µM for Jurkat, NALM6, and REH respectively after 24 hours of treatment. Long-term treatment with NSC305787 reduced the autonomous clonal growth of Jurkat, NALM6, and REH cells in a concentration-dependent manner (p<0.05). Pharmacological inhibition of ezrin also strongly reduced adhesion and migration in ALL cells (all p<0.05). In ALL primary cells, NSC305787 reduced cell viability in an IC50 range from 2.5 to 11.2 µM. NSC305787 (1.6 µM for 24 h), downregulated CCNA2, and upregulated CDKN1A, BCL2L1 and BIM (p<0.05) in Jurkat cells; downregulated CCNA2 and BCL2, and upregulated BCL2L1, BAX, and BAD in NALM6 cells; downregulated CCNA2 and CCNB1 and upregulated CDKN1A, CDKN1B, BCL2L1, MCL1, and BAX in REH cells. In all the cell lines, the inhibitor reduced the phosphorylation of ribosomal protein S6, and induced PARP1 cleavage. Proteomic data from NSC305787-treated (3.2 µM for 12h) NALM6 cells evidenced an upregulation of proteins involved in biological processes like regulation of cell cycle and RNA catabolic processes, and downregulation of mitochondrial translation elongation and translational elongation. Summary/Conclusion: Downregulation of genes related to cell cycle progression (CCNA2 and CCNB1) and anti-apoptotic response (BCL2), and upregulation of genes related to cell cycle arrest (CDKN1A, CDKN1B), pro-apoptotic (BAX) response, support the hypothesis that NSC305787 treatment reduces viability by inducing apoptosis. Our results indicate that the ezrin inhibitor, NSC305787, has antileukemic effects in ALL, including reduction of viability and clonogenicity, anti-invasive effects, and regulation of pathways involved in protein synthesis. Supported by CNPq, CAPES and FAPESP. Keywords: Apoptosis, Acute lymphoblastic leukemia, Rho GTPase, Actin